This research program aims to understand clathrin biochemistry in order to elucidate how clathrin-coated vesicle (CCV) formation and function is regulated in cells. CCVs implement the fundamental membrane traffic pathways of endocytosis and lysosome biogenesis. CCVs also participate in antigen presentation, secretory granule biogenesis and synaptic vesicle regeneration. Thus, CCVs influence cellular pathways that are critical for human health. The characteristic lattice-like CCV coat is formed by interactions between clathrin heavy chains (CHCs). Clathrin light chain (LC) subunits control CCV binding to other proteins. Work from the past funding period generated new hypotheses about how each subunit contributes to CCV function and regulation. Aims 1 and 2 of this proposal focus on the CMC and test the hypothesis that regulation of CCV formation depends on control of key low affinity CHC interactions by protein competition and phosphorylation. CHC interactions with CHC and adaptor proteins will be mapped at the molecular level and the contributions of these interactions to in vitro and in vivo clathrin assembly will be established. The role of CHC phosphorylation in regulating CCV uptake of signaling receptors will also be addressed. Approaches include site-directed mutagenesis of recombinant proteins based on structural information and studying the cellular effects of mutagenesis after siRNA down-regulation and reconstitution. Aims 3 and 4 of this proposal test the hypothesis that clathrin LCs play important regulatory roles at the tissue level. Molecular interactions of LCs with Hip proteins and other binding proteins will be defined through structural and biochemical analysis. The effect of these interactions on cell-substrate contacts, as well as conventional CCV pathways, will be analyzed and their regulation characterized. Experiments to ablate LC gene expression in mice will address LC function in vertebrate embryonic development and in tissue-specific cell lineages, relating molecular information about CCV function to physiological pathways. Public health relevance: The clathrin protein is involved in key pathways that contribute to maintenance of human health including nutrition, lipid metabolism, hormone regulation, response to infection and cell growth control. Understanding clathrin function therefore has relevance for establishing molecular mechanisms of a number of human disease states such as heart disease, diabetes, cancer, neuro-muscular defects and infection and immunity.